Download AmpliTaq Gold® 360 DNA Polymerase Protocol
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® Gold AmpliTaq Polymerase Protocol 360 DNA © Copyright 2009, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by US patent claims and corresponding patent claims outside the US. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as the patented 5’ Nuclease Process claims) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. TRADEMARKS: Applied Biosystems, AB (Design), GeneAmp and Primer Express are registered trademarks and MicroAmp, Veriti and VeriFlex are trademarks of Applied Biosystems Inc. or its subsidiaries in the US and/or certain other countries. AmpliTaq Gold are registered trademarks of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. Part Number 4398943 Rev. B 06/2010 Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi How to use this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii AmpliTaq Gold® 360 DNA Polymerase Protocol . . . . . . . . . . . 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Before you perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Perform PCR using AmpliTaq Gold® 360 DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . . 7 Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Appendix A Ordering Information . . . . . . . . . . . . . . . . . . . . . 13 Materials and equipment not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Appendix B Guidelines for Designing PCR Assays . . . . . . . 17 PCR good laboratory practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Select the amplicon site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Adjust thermal cycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Optimize the PCR conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Appendix C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Chemical hazard warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Chemical safety guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Chemical waste hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Chemical waste safety guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Waste disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Chemical alerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 AmpliTaq Gold® 360 DNA Polymerase Protocol iii Contents Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . 35 Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 How to obtain support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 iv AmpliTaq Gold® 360 DNA Polymerase Protocol Preface This preface covers: Safety information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi How to use this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii AmpliTaq Gold® 360 DNA Polymerase Protocol v Safety information Safety information Note: For general safety information, see this Preface and Appendix C, Safety on page 23. When a hazard symbol and hazard type appear by a chemical name or instrument hazard, see the “Safety” Appendix for the complete alert on the chemical or instrument. Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word—IMPORTANT, CAUTION, WARNING, DANGER—implies a particular level of observation or action, as defined below: IMPORTANT! – Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. CAUTION! – Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. WARNING! – Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. DANGER! – Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. MSDSs The MSDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day. For instructions on obtaining MSDSs, see “Obtaining MSDSs” on page 26. IMPORTANT! For the MSDSs of chemicals not distributed by Applied Biosystems or Ambion, contact the chemical manufacturer. vi AmpliTaq Gold® 360 DNA Polymerase Protocol How to use this guide Purpose of this guide The AmpliTaq Gold® 360 DNA Polymerase Protocol provides all the information you need to perform PCR over a wide range of DNA templates, including some of the most challenging GC-rich sequences. Audience This guide is intended for biologists who have had some experience performing PCR. Assumptions Text conventions This guide assumes that your thermal cycler has been installed by an Applied Biosystems technical representative. This guide uses the following conventions: • Bold text indicates user action. For example: Type 0, then press Enter for each of the remaining fields. • Italic text indicates new or important words and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. • A right arrow symbol () separates successive commands you select from a drop-down or shortcut menu. For example: Select FileOpenSpot Set. Right-click the sample row, then select View Filter View All Runs. User attention words Two user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below: Note: – Provides information that may be of interest or help but is not critical to the use of the product. IMPORTANT! – Provides information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. AmpliTaq Gold® 360 DNA Polymerase Protocol vii How to use this guide viii AmpliTaq Gold® 360 DNA Polymerase Protocol AmpliTaq Gold® 360 DNA Polymerase Protocol This chapter covers: Product information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Before you perform PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Perform PCR using AmpliTaq Gold® 360 DNA Polymerase . . . . . . . . . . . . . . . . . . 7 Analyze the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 AmpliTaq Gold® 360 DNA Polymerase Protocol 1 AmpliTaq Gold® 360 DNA Polymerase Protocol Product information Purpose of the product Use the AmpliTaq Gold® 360 DNA Polymerase to amplify with high specificity a wide range of fragments. About AmpliTaq Gold® 360 DNA Polymerase AmpliTaq Gold® 360 DNA Polymerase, when used with AmpliTaq Gold® 360 Buffer, 10✕ and the optional 360 GC Enhancer, amplifies a wide range of DNA sequence contexts. AmpliTaq Gold® 360 DNA Polymerase is purified by an additional proprietary separation process to reduce contaminating bacterial DNA sequences from the enzyme preparation. This ultra-pure enzyme, in addition to its hot-start capabilities, reduces false positives, amplifies low-level target sequences, and promotes amplification of a variety of templates including those from bacteria and the human genome. The enzyme is quality-control tested to ensure that, in a 5unit aliquot of the enzyme, bacterial 16S ribosomal RNA gene sequences are present in low copy number. About the 360 GC enhancer The 360 GC Enhancer is used for difficult-to-amplify templates, especially for templates with high GC content. You can adjust the amount of 360 GC Enhancer to optimize the PCR reaction. Amplicons that generate nonspecific products may require small amounts of enhancer to improve specificity. Amplicons with high GCcontent require more enhancer. About this protocol This protocol provides: • Procedures and guidelines on PCR using the AmpliTaq Gold® 360 DNA Polymerase • Information on troubleshooting PCR results • A list of equipment and materials required for using the AmpliTaq Gold® 360 DNA Polymerase For more information, refer to the documents shipped with your Applied Biosystems PCR System. Contents The AmpliTaq Gold® 360 DNA Polymerase contains: • • • • AmpliTaq Gold® 360 DNA Polymerase AmpliTaq Gold® 360 Buffer, 10✕ 25 mM Magnesium Chloride 360 GC Enhancer Note: The 360 GC Enhancer, AmpliTaq Gold® 360 Buffer, 10✕, and 25 mM Magnesium Chloride are also available without enzyme. Available kit packaging 2 AmpliTaq Gold® 360 DNA Polymerase is available in the following packaging: AmpliTaq Gold® 360 DNA Polymerase Protocol Product information Quantity Reagents sufficient for 80 × 50-µL reactions: • • • • AmpliTaq Gold 360 DNA Polymerase, 100 U AmpliTaq Gold® 360 Buffer, 10✕, 1 × 1.5 mL 360 GC Enhancer, 1 × 1.5 mL 25 mM MgCl2, 1 × 1.5 mL AmpliTaq Gold 360 DNA Polymerase, 250 U AmpliTaq Gold® 360 Buffer, 10✕, 1 × 1.5 mL 360 GC Enhancer, 1 × 1.5 mL 25 mM MgCl2, 1 × 1.5 mL AmpliTaq Gold 360 DNA Polymerase, 1000 U AmpliTaq Gold® 360 Buffer, 10✕, 4 × 1.5 mL 360 GC Enhancer, 4 × 1.5 mL 25 mM MgCl2, 4 × 1.5 mL AmpliTaq Gold 360 DNA Polymerase, 6 × 250 U AmpliTaq Gold® 360 Buffer, 10✕, 6 × 1.5 mL 360 GC Enhancer, 6 × 1.5 mL 25 mM Magnesium Chloride, 6 × 1.5 mL AmpliTaq Gold 360 DNA Polymerase, 2 × 1500 U AmpliTaq Gold® 360 Buffer, 20✕, 12 × 1.5 mL 360 GC Enhancer, 12 × 1.5 mL 25 mM Magnesium Chloride, 12 × 1.5 mL AmpliTaq Gold 360 DNA Polymerase, 5 × 1000 U AmpliTaq Gold® 360 Buffer, 10✕, 20 × 1.5 mL 360 GC Enhancer, 20 × 1.5 mL 25 mM MgCl2, 20 × 1.5 mL 4398898 ® AmpliTaq Gold 360 DNA Polymerase, 25 × 1000 U AmpliTaq Gold® 360 Buffer, 10✕, 100 × 1.5 mL 360 GC Enhancer, 100 × 1.5 mL 25 mM MgCl2, 100 × 1.5 mL Reagents sufficient for 20,000 × 50-µL reactions: • • • • 4398896 ® Reagents sufficient for 20,000 × 50-µL reactions: • • • • 4398894 ® Reagents sufficient for 4000 × 50-µL reactions: • • • • 4398892 ® Reagents sufficient for 2400 × 50-µL reactions: • • • • 4398833 ® Reagents sufficient for 1200 × 50-µL reactions: • • • • 4398823 ® Reagents sufficient for 800 × 50-µL reactions: • • • • 4398813 ® Reagents sufficient for 200 × 50-µL reactions: • • • • Part number 4398900 ® AmpliTaq Gold 360 DNA Polymerase, 1 × 25,000 U AmpliTaq Gold® 360 Buffer, 10✕, 1 × 150 mL 360 GC Enhancer, 1 × 120 mL 25 mM Magnesium Chloride, 1 × 150 mL AmpliTaq Gold® 360 DNA Polymerase—standalone, 25,000 U, 1 × 5 mL AmpliTaq Gold® 360 DNA Polymerase Protocol 4398843 3 AmpliTaq Gold® 360 DNA Polymerase Protocol Part number Quantity AmpliTaq Gold® 360 Buffer, 10✕ Kit: 4398853 ® • AmpliTaq Gold 360 Buffer, 10✕, 1 × 1.5 mL • 360 GC Enhancer, 1 × 1.5 mL • 25 mM Magnesium Chloride, 1 × 1.5 mL AmpliTaq Gold® 360 Buffer, 10✕ Kit: 4398863 ® • AmpliTaq Gold 360 Buffer, 10✕, 6 × 1.5 mL • 360 GC Enhancer, 6 × 1.5 mL • 25 mM Magnesium Chloride, 6 × 1.5 mL AmpliTaq Gold® 360 Buffer Kit: 4398872 ® • AmpliTaq Gold 360 Buffer, 10✕, 1 × 150 mL • 360 GC Enhancer, 1 × 120 mL • 25 mM Magnesium Chloride, 1 × 150 mL Storage 4 Store the AmpliTaq Gold® 360 DNA Polymerase at − 15 to − 25 °C. AmpliTaq Gold® 360 DNA Polymerase Protocol Workflow Workflow The workflow for PCR using the AmpliTaq Gold® 360 DNA Polymerase: Prepare the reaction mix Prepare the reaction plate or tubes 384-well plate 96-well plate Set up the run method Load and run the plate or tubes Analyze the results AmpliTaq Gold® 360 DNA Polymerase Protocol 5 AmpliTaq Gold® 360 DNA Polymerase Protocol Before you perform PCR Prevent contamination Select an instrument and reaction plate Calculate the number of required reactions Review “PCR good laboratory practices” on page 18. You can perform PCR amplification with any of the instruments listed under “Thermal cyclers” on page 14. Use MicroAmp™ Optical 96-Well Reaction Plates (PN N8010560). Other recommended equipment and consumables are listed beginning on page 14. Calculate the number of reactions to perform for each assay. In your calculations, include extra reactions (approximately one extra reaction for every 10 required reactions) to provide excess volume for the volume lost during reagent transfers. For example, for a 96-well plate, prepare enough volume for approximately 110 reactions. Note: You can run multiple PCRs on one reaction plate. Include controls for each run on the plate. 6 AmpliTaq Gold® 360 DNA Polymerase Protocol Perform PCR using AmpliTaq Gold® 360 DNA Polymerase Perform PCR using AmpliTaq Gold® 360 DNA Polymerase Prepare the reaction mix For the following hazards, see the complete safety alert descriptions in “Specific chemical alerts” on page 29. CHEMICAL HAZARD. AmpliTaq Gold® 360 DNA Polymerase, AmpliTaq Gold 360 Buffer, 10✕. ® IMPORTANT! Avoid generating bubbles when mixing the enzyme. 1. Thaw AmpliTaq Gold® 360 Buffer, 10✕, 25 mM Magnesium Chloride, dNTP mix, primers, template, and (optional) 360 GC Enhancer, then vortex both reagents before use. 2. Mix AmpliTaq Gold® 360 DNA Polymerase by gently pipetting it up and down. 3. Combine the following components in an appropriate tube according to the volumes that are shown in Table 1. Multiply the volume for one reaction component (Table 1) by the total number of reactions, then add that volume to the tube. Table 1 PCR reaction mix Volume per 25-µL reaction (µL) ‡ Volume per 50-µL reaction (µL) PCR-grade water Variable Variable — AmpliTaq Gold® 360 Buffer, 10✕ 2.5 5 1✕ 25 mM Magnesium Chloride 1 to 4 2 to 8 1.0 to 4.0 mM § dNTP mix 2# 4# 200 µM each 1 to 10‡‡ N/A 0.25 1.25 Units per 50 µL reaction §§ Component (Optional)360 GC Enhancer 0.5 to AmpliTaq Gold® 360 DNA Polymerase 0.125 5 ‡‡ Final concentration ‡ If the DNA is difficult to amplify in a 25-µL reaction, performing the PCR in a 50-µL reaction may give better results. § The optimal MgCl2 concentration may vary depending on the primer and template that are used and must be determined by experiment. In most cases, a final concentration of MgCl2 at 2.0 mM in the reaction mix works well. # Contains a 10-mM solution of dNTP (2.5 mM each of dATP, dCTP, dGTP, and dTTP). ‡‡ For targets with 65 to 75% GC, start with 2.5 µL in a 25-µL reaction or 5.0 µL in a 50-µL reaction (10% (v/v) of the reaction). For targets with >75% GC, start with 5 µL in a 25-µL reaction or 10 µL in a 50-µL reaction (20% (v/v) of the reaction). In general, if increased specificity is required, add 0.5. to 1 µL 360 GC Enhancer per 25-µL reaction or add 1 to 2 µL 360 GC Enhancer per 50-µL reaction (2 to 5% (v/v) of the reaction). §§ For some difficult-to-amplify targets, up to 5.0 U per 50 µL of reaction can be added. 4. Cap the tube. 5. Gently vortex the tube on a low setting for no more than 5 seconds to mix the components. AmpliTaq Gold® 360 DNA Polymerase Protocol 7 AmpliTaq Gold® 360 DNA Polymerase Protocol 6. Centrifuge the tube briefly to spin down the contents and to eliminate air bubbles from the solution. 7. Dispense equal volumes of the PCR reaction mix to the reaction plate or into PCR tubes (see Table 1). 8. Place the plate in a MicroAmp™ Splash-Free 96-Well Base or place the tubes in a MicroAmp™ 96-well Base. Keep the plate or tubes in their respective bases throughout the remainder of the protocol. 9. Seal the plate with MicroAmp™ Clear Adhesive Film or cap the tubes with MicroAmp™ 8-Cap Strips. 10. Centrifuge the plate or tubes to collect the liquid at the bottom of the wells. Prepare the reaction plate or tubes 1. Prepare primers and DNA to their appropriate working dilutions (see Table 2). For multiple PCR assays, prepare a master mix of components. 2. With the plate or tubes in the appropriate base, remove the seal from the plate or open the tubes. 3. Add primers and DNA to the appropriate wells or tubes according to Table 2. Include the no-template controls. Table 2 Primer and DNA mix for PCR reactions Volume per 25-µL reaction (µL) ‡ Volume per 50-µL reaction (µL) Primer 1 0.5 to 2.5 1 to 5 0.2 to 1.0 µM § Primer 2 0.5 to 2.5 1 to 5 0.2 to 1.0 µM§ DNA Variable # Variable# <1 µg/reaction ‡‡ Total PCR volume 25 50 — Component Final concentration ‡ If the DNA is difficult to amplify in a 25-µL reaction, performing the PCR in a 50-µL reaction may give better results. § Lowering the primer concentration reduces potential secondary products. # For a no-template control, add an equivalent volume of water. ‡‡ Preferably >104 copies of template but <1 µg DNA/reaction 4. Seal the plate with MicroAmp™ Clear Adhesive Film or cap the tubes with MicroAmp™ 8-Cap Strips. 5. Centrifuge the plate or tubes to collect the liquid at the bottom of the wells or the tubes. Ensure that the wells are uniformly filled. 8 AmpliTaq Gold® 360 DNA Polymerase Protocol Perform PCR using AmpliTaq Gold® 360 DNA Polymerase Set up the run method Set the: • Thermal cycling conditions (see Table 3): Table 3 Three-temperature thermal cycling on a a Veriti™, GeneAmp® PCR System 9700, or 2720 Thermal Cycler Stage Step Temp. Time Holding Activation of AmpliTaq Gold® 360 DNA Polymerase 95 °C 5 to 10 min ‡ Cycling (25 to 40 cycles) Denature 95 °C 15 to 30 sec § Anneal Primer Tm # 30 sec ‡‡ Extend 72 °C 60 sec/kb Holding Final Extension 72 °C 7 min Holding Final hold 4 °C ∞ Gold® ‡ See “Adjusting the hold period for activation” in the AmpliTaq 360 DNA Polymerase Protocol, Appendix B. § For amplicons that are >2 kb, denature for 15 seconds. For amplicons that are ≤2 kb, denature for 30 seconds. # Although any primer can be used with this product, Applied Biosystems recommends using primers with Tms >55 °C. Use the Primer Tm calculator on an Applied Biosystems thermal cycler, or go to www.appliedbiosystems.com/support/techtools/calc. ‡‡ Thirty seconds for denaturation and annealing is adequate when you use Veriti™ or GeneAmp® PCR System thermal cyclers that display a calculated sample temperature. Some models of thermal cyclers may require longer times. • Ramp speed or mode: Standard • Reaction volume (µL): 25 or 50 Load and run the plate or tubes 1. Remove the plate or PCR tubes from the base. 2. Use a MicroAmp™ Optical Film Compression Pad when you use a MicroAmp™ Clear Adhesive Film. 3. Load the reaction plate or tubes into a PCR instrument. 4. Start the run. 5. Unload the reaction plate or tubes after the run is complete. 6. Store the plate or tubes at 4 °C or at − 15 to − 25 °C for long-term storage. For more information Refer to the user guide or getting started guides for your PCR system for more information about setting up the experiment, using and maintaining the instrument, and performing instrument calibrations. AmpliTaq Gold® 360 DNA Polymerase Protocol 9 AmpliTaq Gold® 360 DNA Polymerase Protocol Analyze the results WARNING! CHEMICAL HAZARD. Ethidium bromide. Check the purity of the PCR product Analyze the PCR amplification products by agarose gel electrophoresis. IMPORTANT! To prevent contamination, never bring amplified PCR products into the PCR setup area. 1. Obtain a 1% agarose gel with ethidium bromide stain. You can use a gel of up to 3% agarose with ethidium bromide stain. Set up the electrophoresis apparatus and running buffer according to the manufacturer’s instructions. 2. Add an aliquot of the PCR product to a well of a new plate or to an appropriate new tube. Add an appropriate volume of gel-loading buffer to the PCR-product aliquot. For example, add 1 µL of 10✕ gel-loading buffer to a 9-µL aliquot of PCR reaction. 3. Mix the PCR-product aliquot and buffer in the wells by pipetting up and down, or briefly vortex the samples in the tubes. Spin the plate or pulse-spin the tubes. 4. Dispense the entire volume of the buffer-PCR product aliquot from each well or tube into a well of the gel. 5. Into one well of the gel, load a DNA-ladder marker appropriate to the PCR product length. 6. Run the gel at the voltage or time appropriate to amplicon length and agarose percentage so that the samples run 1/3 to 1/2 the length of the gel. Do not run the dye off the gel. 7. Place the gel on a UV transilluminator. Verify that each lane with a PCR-product aliquot contains one distinct band. For more information 10 Refer to the getting started guides for your PCR system for more information about analyzing a PCR results. AmpliTaq Gold® 360 DNA Polymerase Protocol Troubleshooting Troubleshooting Observation Nonspecific amplification with or without a product band Possible cause Recommended action Carryover contamination • Use the GeneAmp® PCR Carry-Over Prevention Kit (PN N8080068). • Dispose of reagents, make fresh reagents, then repeat the PCR. • Use 1 to 2 µL of 360 GC Enhancer in a 50 µL reaction. Use the 360 GC Enhancer only if there are nonspecific products or products with ≥65% GC (see “Prepare the reaction mix” on page 7). Denaturation temperature is too low or too high Adjust the temperature in increments of 1 degree Celsius. If you have a Veriti™ thermal cycler, adjust the VeriFlex™ Block in increments of 1 degree Celsius for up to six different temperatures. Nonspecific priming Increase the annealing temperature in increments of 1 to 2 degrees Celsius. Primer design is not optimal Review primer design and composition. AmpliTaq Gold® 360 DNA Polymerase Protocol 11 AmpliTaq Gold® 360 DNA Polymerase Protocol Observation Low levels of PCR product or no product band visible Product band is smeared 12 Possible cause Recommended action Template concentration is too low Increase the sample concentration. Experimental sample DNA is damaged or degraded Add more DNA or use sample that has been processed to minimize shearing and nicking. Denaturation time is too short or too long Adjust the time in increments of 5 seconds. Denaturation temperature is too low or too high Adjust the temperature in increments of 1 degree Celsius. If you have a Veriti™ thermal cycler, adjust the VeriFlex™ Block in increments of 1 degree Celsius for up to six different temperatures. Annealing/extension temperature is too high Lower the temperature in increments of 2 degrees Celsius. If you have a Veriti™ thermal cycler, adjust the VeriFlex™ Block in increments of 2 degrees Celsius for up to six different temperatures. Annealing/extension time is too short Increase the time in increments of 15 seconds. Cycle number is too low Increase the cycle number in increments of three cycles. Primer design is not optimal Review primer design and composition. Preincubation/activation time is not sufficient Increase the pre-PCR heat step in increments of 1 minute, or use the “Time Release” protocol (see “Adjusting the hold period for activation” on page 19). Carryover contamination • Use the GeneAmp® PCR Carry-Over Prevention Kit (PN N8080068). • Dispose of reagents, make fresh reagents, then repeat the PCR. Denaturation time is too short or too long Adjust the time in increments of 5 seconds. Denaturation temperature is too low Increase the temperature in increments of 1 degree Celsius. Annealing/extension time is too long Shorten the time in increments of 15 seconds. Cycle number is too high Shorten the cycle number in increments of 3 cycles. Experimental sample DNA is degraded Test a new aliquot of sample. AmpliTaq Gold® 360 DNA Polymerase Protocol Appendix A Ordering Information This appendix covers: Materials and equipment not included . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Thermal cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Other equipment and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 AmpliTaq Gold® 360 Master Mix Protocol 13 Appendix A Ordering Information Materials and equipment not included In addition to the reagents supplied, the items listed in the following table are required: Thermal cyclers Item ‡ Applied Biosystems PN Veriti™ 60-Well Thermal Cycler 4384638 Veriti™ 4375305 96-Well Fast Thermal Cycler Veriti™ 96-Well Thermal Cycler 4375786 Veriti™ 384-Well Thermal Cycler 4388444 2720 Thermal Cycler 4359659 Aluminum 96-Well GeneAmp® PCR System 9700, Sample Block Module 4314445 Gold-plated 96-Well GeneAmp® PCR System 9700 4314878 ‡ Only one thermal cycler or one PCR system is required. Other equipment and consumables 14 Item Source MicroAmp™ Optical 96-Well Reaction Plates Applied Biosystems (PN N8010560) MicroAmp™ Splash-Free 96-Well Base Applied Biosystems 4312063 MicroAmp™ 8-Tube Strip, 0.2 mL Applied Biosystems (PN N8010580) MicroAmp™ 8-Cap Strip Applied Biosystems (PN N8010535) MicroAmp™ 96-well Base Applied Biosystems N8010531 dNTP Mix Applied Biosystems (PN N8080260) MicroAmp™ 96-Well Tray/Retainer Set, 10 sets Applied Biosystems (PN 403081) Nuclease-free water (not DEPC-treated), 500 mL Applied Biosystems (PN AM9930) MicroAmp™ Clear Adhesive Film Applied Biosystems (PN 4306311) MicroAmp ™ Optical Film Compression Pad ‡. Applied Biosystems (PN4312639 Centrifuge with plate adapter Major laboratory supplier (MLS) § AmpliTaq Gold® 360 Master Mix Protocol Materials and equipment not included Item Source Pre-cast agarose gels, 1% up to 3% with ethidium bromide stain MLS Agarose MLS Disposable gloves MLS Electrophoresis apparatus MLS Microcentrifuge MLS Pipettes, positive-displacement or airdisplacement MLS Pipette tips with filter plugs MLS Polypropylene tubes MLS TBE buffer MLS TE buffer MLS Vortex MLS Disposable gloves MLS Microcentrifuge MLS 1.5-mL microcentrifuge tubes MLS Tris-EDTA (TE) buffer, pH 8.0 MLS Vortexer MLS ‡ See instrument manual for compatibility. § For the MSDS of any chemical not distributed by Applied Biosystems, contact the chemical manufacturer. Before handling any chemicals, refer to the MSDS provided by the manufacturer, and observe all relevant precautions. For more product recommendations, visit the PCR technology page at: www3.appliedbiosystems.com/applicationstechnologies/PCR/index.htm?newGl obalNav=true AmpliTaq Gold® 360 Master Mix Protocol 15 Appendix A Ordering Information 16 AmpliTaq Gold® 360 Master Mix Protocol Appendix B Guidelines for Designing PCR Assays This appendix covers: PCR good laboratory practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Select the amplicon site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Adjust thermal cycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Optimize the PCR conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 AmpliTaq Gold® 360 Master Mix Protocol 17 Appendix B Guidelines for Designing PCR Assays PCR good laboratory practices General PCR practices 18 When preparing samples for PCR amplification: • Use a positive-displacement pipette or aerosol-resistant pipette tips. • Follow proper pipette-operating techniques to prevent aerosols. • Wear clean gloves and a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation). • Change gloves whenever you suspect that they are contaminated. • Maintain separate areas and dedicated equipment and supplies for: – Sample preparation – PCR setup – PCR amplification – Analysis of PCR products • Never bring amplified PCR products into the PCR setup area. • Open and close all sample tubes carefully. Try not to splash or spray PCR samples. • Keep reactions and components capped as much as possible. • Clean lab benches and equipment periodically with 10% bleach solution. Use DNAZap™ Solution (PN AM9890). AmpliTaq Gold® 360 Master Mix Protocol Select the amplicon site Select the amplicon site Using Primer Express® Software, select an amplicon site within the target sequence (refer to the Primer Express® Version 3.0 Getting Started Guide and Software Help). Guidelines • Design primer pairs according to Primer Express Software guidelines. • Use a primer pair that is specific to the target gene and does not amplify pseudogenes or other related genes. • Test the primer pairs, then select the primer pair that produces the largest amount of specific product and the least amount of non-specific product. To determine specific product, compare migration through an agarose gel of amplicons to that of the DNA bands of known length in a DNA ladder. Adjust thermal cycling Adjusting the hold period for activation For general PCR runs, Applied Biosystems recommends a pre-PCR activation setup of 95 °C for 10 minutes. Perform a titration of pre-PCR activation times (2 to 10 minutes in 1-minute intervals) to find the best enzyme activity for your reaction. You can also activate AmpliTaq Gold® 360 DNA Polymerase to release active enzyme slowly over time (time release). Time release allows enzyme activity to increase with cycle number as the amount of template increases. This type of PCR can be accomplished • With limited activation during the pre-PCR hold period or • Without activation during the pre-PCR hold period In a “time-release” protocol, the enzyme is released slowly so that the enzyme concentration matches the template concentration, thereby increasing enzyme specificity. When no or limited (1-to-2 minute) pre-PCR activation is used, the enzyme is released gradually during the denaturation step (95 °C for 15 to 30 seconds) of each cycle. Because the enzyme is released slowly, up to 5 additional cycles may be required. Limiting the amount of active enzyme at the beginning of the amplification reaction enhances specificity in the early PCR cycles. Adjusting denaturation conditions • In the early cycles, make sure that your DNA template is completely denatured. • Do not exceed a denaturation temperature of 95 to 96 °C (Gelfand and White, 1990). • A denaturation period of 30 seconds is adequate when using Veriti™ and GeneAmp® PCR System thermal cyclers with a calculated in-tube temperature. Some models of thermal cyclers may require longer denaturation times. Adjusting annealing conditions • For increased product specificity, use annealing temperatures higher than 45 °C (Saiki, Gelfand, and Stoffel, 1988; Rychlik, Spencer, and Rhoads, 1990). AmpliTaq Gold® 360 Master Mix Protocol 19 Appendix B Guidelines for Designing PCR Assays • Determine the optimum annealing temperature by testing at increments of 5 or fewer degrees Celsius until the maximum specificity is reached. • Computer programs that calculate primer melting temperatures (Tm) can help you narrow the range of annealing temperatures to test. For such a Tm calculator, go to http://www.appliedbiosystems.com, then select Services & Support Technical Tools Tm Calculator. The GeneAmp® PCR System 9700 Thermal Cycler also contains a Tm calculator. • Thirty seconds is adequate annealing time when using the Veriti™ and GeneAmp PCR System thermal cyclers with a calculated in-tube temperature. Some models of thermal cyclers may require longer annealing times. Adjusting extension conditions • The length of the target sequence affects the required extension time. Longer targets require increased extension times. In general, allow an extension time of approximately 60 seconds per 1000 bases at 72 °C. • As the amount of DNA increases, the number of DNA polymerase molecules may become limiting. Compensate for this limitation by increasing the extension time in later cycles. Optimize the PCR conditions Optimizing template concentration Optimizing enhancer concentration • The DNA segment to be amplified from the template can be up to 4 kb long (Jeffreys et al., 1988), although 100 to 1000 bases are more typical and easier to amplify. • Start with enough copies of the template to obtain a signal after 25 to 30 cycles. More than 10 4 copies but less than 1 µg of human genomic DNA per 50-µL reaction is the recommended range. • If the target DNA concentration is low, you may need more than 35 cycles to produce sufficient product for analysis. As few as 1 to 10 target copies can be amplified (Saiki, Gelfand, Stoffel, 1988; Chou et al., 1992). Validation for lowcopy-number amplifications is best done for an average of 5 to 10 target molecules per sample to avoid statistical false negatives. The 360 GC Enhancer helps amplify amplicons that are difficult to amplify, including amplicons that are GC-rich or have GC-repeats or that generate nonspecific products. In a 50-µL reaction, for targets with: • 65 to 75% GC, start with 5 µL. • >75% GC, start with 10 µL. In general, if increased specificity is required, add 1 to 2 µL per 50-µL reaction. The 360 GC Enhancer can reduce nonspecific amplification and improve the yield of specific products. However, excessive use of the 360 GC Enhancer can reduce yield, particularly for non-GC-rich amplicons. 20 AmpliTaq Gold® 360 Master Mix Protocol Optimize the PCR conditions Optimizing Magnesium Chloride concentration The magnesium ion concentration that is required for optimal PCR amplification depends on the specific set of primers and template. Too much or too little MgCl2 reduces amplification efficiency or results in amplification of non-target sequences. The optimal MgCl2 concentration must be determined by experiment. To determine the optimum MgCl2 concentration for each primer set: • Use the supplied 25 mM MgCl2 to adjust the magnesium ion concentration. • Vary the concentration of MgCl2 around 1.8 mM. A typical range is 1.0 to 4.0 mM. • Raise the MgCl 2 concentration in the reaction mix proportionately if the samples contain EDTA, citrate, or other chelators. • Adjust the MgCl 2 concentration in parallel with significant changes in the concentration (higher or lower) of sample DNA or dNTPs to keep the free magnesium ion concentration constant. For example, reduce the concentration of dNTP from 200 µM each to 40 µM each, and reduce the MgCl 2 concentration from the higher concentration to 640 µM. Optimizing dNTP concentration Optimizing enzyme concentration The dNTP concentration that is recommended for PCR is 200 µM for each dNTP. If the blend of dNTPS is changed, and the concentration of any one dNTP is significantly different from the other dNTPs, then AmpliTaq Gold® 360 DNA Polymerase tends to misincorporate, slow down, and/or terminate prematurely (Innis et al., 1988). Lower concentrations of dNTPs (40 µM) tend to promote polymerase fidelity (Eckert and Kunkel, 1992). For difficult-to-amplify targets, increasing the AmpliTaq Gold® 360 DNA Polymerase up to 5 U per 50 µL reaction may improve the yield. AmpliTaq Gold® 360 Master Mix Protocol 21 Appendix B Guidelines for Designing PCR Assays 22 AmpliTaq Gold® 360 Master Mix Protocol Appendix C Safety This appendix covers: Chemical hazard warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Chemical safety guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 MSDSs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Chemical waste hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Chemical waste safety guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Waste disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Chemical alerts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 AmpliTaq Gold® 360 DNA Polymerase Protocol 23 Appendix C Safety Chemical hazard warnings WARNING! CHEMICAL HAZARD. Before handling any chemicals, refer to the Material Safety Data Sheet (MSDS) provided by the manufacturer, and observe all relevant precautions. WARNING! CHEMICAL HAZARD. All chemicals in the instrument, including liquid in the lines, are potentially hazardous. Always determine what chemicals have been used in the instrument before changing reagents or instrument components. Wear appropriate eyewear, protective clothing, and gloves when working on the instrument. WARNING! CHEMICAL HAZARD. Four-liter reagent and waste bottles can crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. 24 AmpliTaq Gold® 360 DNA Polymerase Protocol Chemical safety guidelines Chemical safety guidelines To minimize the hazards of chemicals: • Read and understand the Material Safety Data Sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See “About MSDSs” on page 26.) • Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. • Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS. • Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer’s cleanup procedures as recommended in the MSDS. • Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. AmpliTaq Gold® 360 DNA Polymerase Protocol 25 Appendix C Safety MSDSs About MSDSs Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with shipments of hazardous chemicals to new customers. They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated. MSDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files. Obtaining MSDSs The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain MSDSs: 1. Go to www.appliedbiosystems.com, click Support, then select MSDS. 2. In the Keyword Search field, enter the chemical name, product name, MSDS part number, or other information that appears in the MSDS of interest. Select the language of your choice, then click Search. 3. Find the document of interest, right-click the document title, then select any of the following: • Open – To view the document • Print Target – To print the document • Save Target As – To download a PDF version of the document to a destination that you choose Note: For the MSDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer. Chemical waste hazards CAUTION! HAZARDOUS WASTE. Refer to Material Safety Data Sheets and local regulations for handling and disposal. WARNING! CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury, illness, or death. WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. 26 AmpliTaq Gold® 360 DNA Polymerase Protocol Chemical waste safety guidelines Chemical waste safety guidelines To minimize the hazards of chemical waste: • Read and understand the Material Safety Data Sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. • Provide primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) • Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. • Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS. • Handle chemical wastes in a fume hood. • After emptying a waste container, seal it with the cap provided. • Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. Waste disposal If potentially hazardous waste is generated when you operate the instrument, you must: • Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. • Ensure the health and safety of all personnel in your laboratory. • Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. AmpliTaq Gold® 360 DNA Polymerase Protocol 27 Appendix C Safety Biological hazard safety General biohazard warning WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: • U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no. 017-04000547-4; bmbl.od.nih.gov) • Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR§1910.1030; www.access.gpo.gov/ nara/cfr/waisidx_01/29cfr1910a_01.html). • Your company’s/institution’s Biosafety Program protocols for working with/handling potentially infectious materials. Additional information about biohazard guidelines is available at: www.cdc.gov 28 AmpliTaq Gold® 360 DNA Polymerase Protocol Chemical alerts Chemical alerts General alerts for all chemicals Specific chemical alerts May cause eye, skin, and respiratory tract irritation. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. CHEMICAL HAZARD. AmpliTaq Gold® 360 DNA Polymerase. CHEMICAL HAZARD. AmpliTaq Gold® 360 Buffer, 10✕. WARNING! CHEMICAL HAZARD. Ethidium bromide causes eye, skin, and respiratory tract irritation and is a known mutagen (that is, it can change genetic material in a living cell and has the potential to cause cancer). AmpliTaq Gold® 360 DNA Polymerase Protocol 29 Appendix C Safety 30 AmpliTaq Gold® 360 DNA Polymerase Protocol Bibliography Birch, D. E., Kolomodin, L., Laird, W. J., McKinney, N., Wong, J., Young, K. K. Y., Zangenberg, G. A., and Zoccoli, M. A. 1996. Simplified Hot Start PCR. Product review in Nature 381: 445-446. Bost, D. A., Zalloua, P., and Abramson, R. D. 1997. AmpliTaq Gold: Biochemical Characterization and PCR Optimization. The FASEB Journal. 11: A1370. Chou, Q., Russell, M., Birch, D.E., Raymond, J., and Bloch, W. 1992. Prevention of pre-PCR mis-priming and primer dimerization improves low- copy-number amplifications. Nucleic Acids Res. 20:1717–1723. Eckert, K.A. and Kunkel, T.A. 1992. The fidelity of DNA polymerases used in the polymerase chain reactions. In: PCR: A Practical Approach. McPherson, M.J., Quirke, P., and Taylor, G.R., eds. New York: Oxford University Press. 225–244. Faloona, F., Weiss, S., Ferre, F., and Mullis, K. 1990. Direct detection of HIV sequences in blood high-gain polymerase chain reaction [abstract]. In: 6th International Conference on AIDS, University of California, San Francisco: San Francisco (CA). Abstract 1019. Gelfand, D.H. and White, T.J. 1990. Thermostable DNA polymerases. In: PCR Protocols: A Guide to Methods and Applications. Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds. San Diego: Academic Press. 129–141. Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5´→3´ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88:7276–7280. Innis, M.A., Myambo, K.B., Gelfand, D.H., and Brow, M.A. 1988. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85:9436–9440. Jeffreys, A.J., Wilson, V., Neumann, R., and Keyte, J., 1988. Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells. Nucleic Acids Res. 16:10953-10971. Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237–238. Kwok, S. 1990. Procedures to minimize PCR-product carry-over. In: PCR Protocols: A Guide to Methods and Applications. Innis, M.A., Gefland, D.H., Sninsky, J.J., and White, T.J., eds. San Diego: Academic Press. 142–145. Mullis, K.B. and Faloona, F.A., 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology 155:335–350. AmpliTaq Gold® 360 DNA Polymerase Protocol 31 Bibliography Lawyer, F.C., Stoffel, S., Saiki, R.K., Myambo, K., Drummond, R., and Gelfand, D.H. 1989. Isolation, characterization, and expression in E. coli of the DNA polymerase gene from the extreme thermophile, Thermus aquaticus, J. Biol. Chem. 264:6427-6437. Richardson, C.C. 1966. DNA polymerase from Escherichia coli. In: Procedures in Nucleic Acid Research. Cantoni, G.L. and Davies, D.R., eds. New York: Harper & Row. 263–276. Rychlik, W., Spencer, W.J., and Rhoads, R.E. 1990. Optimization of the annealing temperature for DNA amplification in vitro [published erratum appears in Nucleic Acids Res 1991 Feb 11;19(3):698]. Nucleic Acids Res. 18:6409–6412. Saiki, R.K., Gelfand, D.H., Stoffel, S., etþal. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491. Saiki, R.K., Scharf, S.J., et al., 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354. 32 AmpliTaq Gold® 360 DNA Polymerase Protocol Glossary amplicon A segment of DNA amplified during PCR. biological replicates Reactions that contain identical components and volumes, but evaluate separate samples of the same biological source (for example, multiple samples of the same liver tissue). See also technical replicates. cycling stage In the thermal profile, a stage that is repeated. A cycling stage is also called an amplification stage. diluent A reagent used to dilute a sample or standard before it is added to the PCR reaction. The diluent can be buffer or water. forward primer Oligonucleotide that flanks the 5′ end of the amplicon. The reverse primer and the forward primer are used together in PCR reactions to amplify the target. holding stage In the thermal profile, a stage that can include one or more steps. You can add a holding stage to the thermal profile to activate enzymes, to inactivate enzymes, or to incubate a reaction. melting temperature (Tm) In melt-curve experiments, the temperature at which 50% of the DNA is doublestranded and 50% of the DNA is dissociated into single-stranded DNA. The Tm is displayed in the melt curve. mode See ramp speed. negative control (NC) The task for targets in wells that contain water or buffer instead of sample. No amplification of the target should occur in negative control wells. Also called no template control (NTC). no-template control (NTC) See negative control (NC). quantity In quantitation experiments, the amount of target in the samples. Absolute quantity can refer to copy number, mass, molarity, or viral load. Relative quantity refers to the fold-difference between normalized quantity of target in the sample and normalized quantity of target in the reference sample. ramp The rate at which the temperature changes during the instrument run. The ramp is defined as a percentage. The ramp for the melt curve step is defined as a temperature increment. In the graphical view of the thermal profile, the ramp is indicated by a diagonal line. AmpliTaq Gold® 360 DNA Polymerase Protocol 33 Glossary ramp speed Speed at which the temperature ramp occurs during the instrument run. For optimal results using the AmpliTaq Gold® 360 DNA Polymerase, Applied Biosystems recommends using the standard ramp speed. reaction mix A solution that contains all components to run the PCR reaction, except for the template (sample, standard, or control). replicate group A set of identical reactions in an experiment. replicates See biological replicates or technical replicates. reverse primer An oligonucleotide that flanks the 3′ end of the amplicon. The reverse primer and the forward primer are used together in PCR reactions to amplify the target. reverse transcriptase An enzyme that converts RNA to cDNA. Reverse transcriptase is added to the PCR reaction to perform 1-step RT-PCR. run method Definition of the reaction volume and the thermal profile for the instrument run. sample The template that you are testing. stage In the thermal profile, a group of one or more steps. In core PCR, there are two types of stages: holding stage (including pre-PCR read and post-PCR read) and cycling stage (also called amplification stage). step A component of the thermal profile. For each step in the thermal profile, you can set the ramp rate (ramp increment for melt-curve steps), hold temperature, and hold time (duration). target The nucleic acid sequence that you want to amplify and detect. technical replicates Identical reactions that contain identical components and volumes and that evaluate the same sample. See also biological replicates. thermal profile Part of the run method that specifies the temperature, time, and ramp for all steps and stages of the PCR instrument run. Tm See melting temperature (Tm). 34 AmpliTaq Gold® 360 DNA Polymerase Protocol Documentation and Support Related documentation You can download the following documents and other product-support documents from: http://docs.appliedbiosystems.com/search.taf Document Part number AmpliTaq Gold® 360 DNA Polymerase Product Insert 4398303 AmpliTaq® 360 DNA Polymerase Quick Reference Card 4398953 Applied Biosystems Veriti™ Thermal Cycler User Guide 4375799 GeneAmp® PCR System 9700 Base Module User’s Manual 4303481 GeneAmp® PCR System 9700 96Well Sample Block Module 4316011 AmpliTaq Gold® 360 DNA Polymerase Protocol 35 Documentation and Support How to obtain support For the latest services and support information for all locations, go to www.appliedbiosystems.com, then click the link for Support. At the Support page, you can: • Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. • Search through frequently asked questions (FAQs) • Submit a question directly to Technical Support • Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents • Download PDF documents • Obtain information about customer training • Download software updates and patches Send us your comments Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can e-mail your comments to: [email protected] IMPORTANT! The e-mail address above is for submitting comments and suggestions relating only to documentation. To order documents, download PDF files, or for help with a technical question, see “How to obtain support” above. 36 AmpliTaq Gold® 360 DNA Polymerase Protocol Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at www.appliedbiosystems.com. Applied Biosystems is committed to providing the world’s leading technology and information for life scientists. Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free (In North America): +1 800.345.5224 Fax: +1 650.638.5884 06/2010 www.appliedbiosystems.com Part Number 4398943 Rev. B